Formulation of ZnPcs

Phototoxicity and fluorescence of Pcs is mostly hampered by their low water-solubility and tendency to aggregate. Their incorporation into suitable and biodegrable formulations is commonly accepted as a necessity in the development of new therapeutics. Indeed, development of drug delivery systems such as liposomes, micelles and nanoparticles could improve the unfavorable biodistribution of free Pcs (i.e., improvement of PS pharmacokinetic properties, better targeting of diseased tissues due to size of the particles, association to serum proteins and specific activation of the PS through localized delivery of the PS) as well as avoidance of aggregation and loss of phototoxic activity/fluorescence which should result in a better therapeutic outcome (see following paragraphs).

Zinc based phthalocyanines have been mostly evaluated as liposomal formulations.

Liposomes are phospholipids vesicles that present the advantage to entrap either hydrophilic drugs in the core of the phospholipid vesicle or hydrophobic drugs within the lipid membrane.

Incorporation of drugs into liposomes results in passive targeting of tumors^159,160. In studies reported in this review, only small unilamellar vesicles (i.e., one phospholipid bilayer) were used.

Liposomal formulation of ZnPc showed photocytotoxic effect in vitro on a large variety of tumor cell lines^161-163. The drug CGP55847 commercialized by CIBA has been the first Pc reaching clinical trials although no clinical results have been reported. Ben-Hur and Chan reported that the “early phase I/II clinical studies […] were discontinued but not for medical reasons”¹⁶⁴.

In vivo, ZnPc being incorporated in dipalmitoyl-phosphatidylcholine (DPPC) liposomes showed complete response at drug doses as low as 0.14 mg/kg in mice inoculated with MS-2

48 fibrosarcoma. At a slightly lower dose of 0.12 mg/kg (i.v.), ZnPc was found to be “associated exclusively with lipoprotein fraction”, i.e., whether it was complexed with low density lipoproteins (LDL) or incorporated into liposomal structures, and 70% of the drug was cleared from the body within 12 h independently from its carrier system, while the remaining ZnPc was slowly eliminated. However, ZnPc associated with LDL presented a higher selectivity toward tumor than ZnPc liposomes with a to-liver ratio of 2.27 and 1.04 and a tumor-to-muscle ratio of 4.20 and 3.84 after 24 h post-injection, respectively. Nonetheless, the selectivity was somewhat limited by the redistribution of the photosensitizer among other lipoproteins, especially high density lipoproteins. As liposomal formulations of benzoporphyrin derivative, liposomal CGP55847 seems to be transferred to serum lipoproteins and more predominantly to low density lipoproteins^165-169 explaining the propensity of the PS to selectively accumulate in tumors^163,170. Rodal et al. suggested that the observed internalization of ZnPc was not following the endocytotic pathway of LDL but rather occurred via diffusion through the cellular membrane after binding to the LDL receptor¹⁶¹.

In vivo studies on different liposomal formulations consisting of a mixture 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (OOPS) revealed that intratumoral distribution pattern of ZnPc in C57BL/6 mice bearing Ehrlich carcinomas or B16 melanomas was a time dependent process¹⁷¹. In agreement with van Leengoed et al.¹⁷², 3 h after intravenous injection of 0.5 mg/kg of ZnPc, the photosensitizer was present in and around the tumor vasculature but not 24 h post-injection. Indeed, using a dorsal skinfold chamber model, van Leengoed et al.¹⁷², could detect vascular occlusion within five minutes after photoactivation which resolved 30 min after irradiation. Moreover, maximal vasculature damage and tumor-to-muscle ratio from 8:1 to 14:1 have been reported for this liposomal delivery system¹⁷³.

Nanoparticles are another type of drug delivery system that enables the incorporation of higher drug doses of hydrophobic drugs. Passive targeting of tumors is also achieved due to their size (i.e., below 1,000 nm, generally 200 nm) through EPR effect. A strategy to increase the circulation time of the nanoparticles as well as other drug delivery systems is the incorporation of PEG moieties on their surface. It is commonly accepted that PEGylation creates hydrophilic barrier that reduces the immunogenicity of the drug delivery system and lowers its clearance from the body^174-176.

49 Polylactic acid (PLA) based nanoparticular formulations of ZnPc were investigated by Allémann et al.^135,136. The coating of nanoparticles with PEG moieties resulted in the decrease of the reticuloendothelial system and an increase in the PS tumor retention¹³⁵. Incorporation of 25 (Figure 10) into PEG coated nanoparticles or using a Cremophor EL based emulsion increased the bioavailability as compared to uncoated-NP by a factor of four in BALB/c mice bearing EMT-6 tumors. The tumor-to-skin and tumor-to-muscle ratios for the PEG-NP and Cremophor based formulation were 2 to 21, respectively, with a maximum concentration in tumor 48 h post-injection¹³⁵. However, the nanoparticular drug delivery system was nearly five times more efficient for PDT-mediated treatment of mice bearing EMT-6 tumors¹³⁶. This is in agreement with a study reported by Fadel et al.¹⁷⁷ describing that tumor bearing animals treated with ZnPcs laden poly(lactic-coglycolic acid) nanoparticles showed the best PDT outcome, highest tumor growth delay and longest survival times as compared to mice treated with the free PS.

An interesting approach was recently described by Kataoka and co-workers^178-181 where dendrimer based zinc phthalocyanines showed promising results in PDT and PCI applications.

Indeed, they could manage to prepare anionic dendrimer zinc phthalocyanine (DPc) incorporated into positively charged poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) block copolymeric micelles referred to as polyion complex micelles (PIC)¹⁸⁰. The substitution of large dendritic parts avoids the aggregation and increases their water solubility¹⁸¹. These micelles were tested both in vitro on human lung adenocarcinoma cells A549 and in vivo on (Balb/c nu/nu) mice bearing A549 tumors. It was shown that 7.6 times more of encapsulated DPc in micelles (DPc/m) was taken up and that these are 78 times more phototoxic in vitro than free DPc. In addition, subsequent in vivo studies confirmed the benefits of micelle encapsulation of DPc by delayed tumor growth and limited skin photosensitization.

Another interesting approach for drug delivery of hydrophobic PS is their conjugation to cyclodextrins. This strategy enables the water-solubilisation of lipophilic PS and thus its systemic administration at high concentrations. In one report, Baugh and al. proceeded to the conjugation of zinc based phthalocyanines to cyclodextrins. The double carbon bounding linkage between the dimers was cleaved, upon light activation and in presence of oxygen;

resulting in the release of the Pc. Hence, this approach could be a promising systemic carrier of hydrophobic PS for photodynamic therapy^182,183.

Formulation of ZnPcs in gels or (micro)emulsions could be of interest in topical application.

Indeed, several studies reported improved photophysical and aggregation properties of

50 ZnPcs^184,185 as well as successful delivery to the skin of sulfonated ZnPc either via simple skin penetration¹⁸⁴ or through iontophoresis¹⁸⁶.

2.3. Silicon Based Phthalocyanines: Pc 4 and its Analogues 2.3.1. SiPc-SAR

Several silicon based phthalocyanines (SiPc) mostly aiming at revealing the influence of axial ligands have been proposed and tested both in vitro and in vivo. Probably, the most famous representative of these compounds with some commercial value is “La Jolla Blue”. Water-solubility of this dye absorbing at 680 nm as well as prevention of aggregation is provided by two axial polyethylene oxide moieties. Then, two peripherial carboxy groups at the macrocycle can be used for the coupling to biological molecules such as antibodies. This has been used for the design of an antibody for an FDA cleared in vitro immunofluorescence assay.

It has been shown by He et al.¹⁸⁷, that silicon based phthalocyanines with short aminosiloxy ligands [49 (Pc 4) and 51] are more phototoxic effect in Chinese hamster lung fibroblasts V79 and Murine leukemic lymphoblasts L5178Y-R as compared to compounds such as 50 and 52 with longer axial ligands (see Figure 17).

51 Figure 17. Chemical structure of Pc 4 (49) and derivatives 50–58^187-194

52 In another report¹⁹⁴, the PDT effects of these compounds in vivo on C3H/HeN mice bearing RIF-1 tumors were examined. The phthalocyanines were solubilized in a Cremophor EL emulsion and light was given 24 h post Pc injection. Except 52, all axially substituted Si-Pc resulted in a complete remission. The authors suggested that 51-mediated PDT may induce cellular destruction via a slower mechanism than Pc 4-mediated PDT.

In 2009, Rodriguez et al.¹⁸⁸, compared Pc 4 to compounds 51, 53, 54 and 55 on the human breast cancer cell line MCF-7c3. These analogues showed, among other features, higher cellular uptake and phototoxicity than Pc 4. Biaxial substitution reduced aggregation which, in turn, can explain their higher photodynamic efficiency. Furthermore, the presence of a hydroxyl group at one side seems to enhance the phototoxicity of the SiPcs. However, as shown by confocal microscopy, except for 51, Pc 4 analogues seem to act through a different destructive pathway due to their principle association with lysosomes rather than with mitochondria/endoplasmic reticulum. Interestingly, when clonogenic assays were performed 51 and Pc 4 had a similar behavior. In contrast, 51 exhibited a 4-fold lower IC50 in comparison to Pc 4 (49) as demonstrated by means of a MTT test. Using bi-axially substituted polyamine SiPcs with fluorescence quantum yields in aqueous media of 0.12–0.21, IC50

ranging from 450 to1 nM have been reported in HT29 cells by Ng and co-workers¹⁹⁵.

Moreover, the same group of research developed several SiPcs substituted axially with β-cyclodextrins^196-198. They could reach a photocytotoxic effect on human colon adenocarcinoma and hepatocarcinoma cells (HT29 and HepG2 respectively) with reported IC50 ranging from 21 nM to 1.32 μM. In addition, early in vivo studies conducted by Lau et al.¹⁹⁷ show promising results as drug doses as low as 1 μmol/kg for a light dose of 30 J/cm² are efficient enough to suppress tumor growth in mice bearin HT29 xenografts.

Another promising approach used by the same group is the glucoconjugation of SiPcs^190,199. In their report, Chan et al.¹⁹⁹ reported IC50 values as low as 6 nM on the same cell lines and could “retard tumor growth” in the same animal model.

Pc 4 has been developed at Case Western Reserve University at the beginning of the 90s.

Since then Pc 4-mediated PDT has been reported effective in vitro against various tumor cell lines of different origin187,200-205. In experimental animal models for ovarian and colon cancer, no photodynamic effect was observed with doses of 0.4 mg/kg²⁰², whereas with doses of 0.6 or 1 mg/kg complete remission or at least significant tumor volume reduction occurred

53 between 3 to 7 days post-PDT. Furthermore, Pc 4-PDT was associated with a delay of tumor regrowth from 9 ²⁰³ up to 90 days²⁰².

A recent study conducted on immunideficient mice bearing papillomas²⁰⁶ induced by the administration of cottontail rabbit papillomavirus led to the conclusion that Pc 4 at a dose of 1mg/kg and at a light dose of 150 J/cm² given 48 h administration resulted in complete remission in 87% of the cases with no observable re-growth during 79 days. In addition, it was suggested that the rapid destruction observed in Pc 4-mediated PDT could be explained by the mitochondrial/endoplasmic reticulum¹⁸⁸ localization of Pc 4 mainly leading to apoptosis and necrosis^207-209. However in some cases vascular occlusion was also reported¹⁸⁸. Moreover, Anderson et al.²¹⁰ demonstrated that Pc 4 exhibited less skin photosensititization as compared to Photofrin®. Topically applied, Oleinick and co-workers established that Pc 4 is

“effectively delivered into the human skin”²¹¹ and was consequently investigated clinically. In these trials, Pc 4-mediated PDT was reported to be well tolerated in patients and could have promising application in mycosis fungoides treatment²¹².

Recently, Fong and coworkers reported the synthesis and characterization of several silicon-based phthalocyanines189,213-217. Out of this series, BAM-SiPc 56 (see Figure 17) having an IC50 as low as 0.015 μM on HepG2, Hep3B, HT29 and J744 was the most potent. However, this compound was phototoxic on normal liver cells (i.e., WRL-68 with IC50 of 0.035 microM under the same experimental conditions)189,192,218. In nude mice bearing hepatocarcinoma HepG2 and colorectal adenocarcinoma HT29 tumors the same compound showed remission/regression and tumor growth delay 15 days after PDT at a drug dose of 1μmol/kg¹⁹¹.

Barge et al.¹⁹³ could also covalently link axial cholesterol moiety (Chol-O-SiPc) (Figure 17) and reported a seven fold higher potency of this derivative as compared to AlClPc (with IC50

of approximatively 8 nM) in vitro, on the pigmented melanoma cell lines M3Dau and SK-MEL-2.